Alpha-Deoxyguanosine to Reshape the Alpha-Thrombin Binding Aptamer.

Modification of DNA aptamers is aimed at increasing their thermodynamic stability, and improving affinity and resistance to biodegradation. G-quadruplex DNA aptamers are a family of affinity ligands that form non-canonical DNA assemblies based on a G-tetrads stack. Modification of the quadruplex core is challenging since it can cause complete loss of affinity of the aptamer. On the other hand, increased thermodynamic stability could be a worthy reward. In the current paper, we developed new three- and four-layer modified analogues of the thrombin binding aptamer with high thermal stability, which retain anticoagulant activity against alpha-thrombin. In the modified aptamers, one or two G-tetrads contained non-natural anti-preferred alpha-deoxyguanosines at specific positions. The use of this nucleotide analogue made it possible to control the topology of the modified structures. Due to the presence of non-natural tetrads, we observed some decrease in the anticoagulant activity of the modified aptamers compared to the natural prototype. This negative effect was completely compensated by conjugation of the aptamers with optimized tripeptide sequences.

The Regioselective Conjugation of the 15-nt Thrombin Aptamer with an Optimized Tripeptide Sequence Greatly Increases the Anticoagulant Activity of the Aptamer.

Currently, oligonucleotide therapy has emerged as a new paradigm in the treatment of human diseases. In many cases, however, therapeutic oligonucleotides cannot be used directly without modification. Chemical modification or the conjugation of therapeutic oligonucleotides is required to increase their stability or specificity, improve their affinity or inhibitory characteristics, and address delivery issues. Recently, we proposed a conjugation strategy for a 15-nt G-quadruplex thrombin aptamer aimed at extending the recognition interface of the aptamer. In particular, we have prepared a series of designer peptide conjugates of the thrombin aptamer, showing improved anticoagulant activity. Herein, we report a new series of aptamer-peptide conjugates with optimized peptide sequences. The anti-thrombotic activity of aptamer conjugates was notably improved. The lead conjugate, TBA-GLE, was able to inhibit thrombin-induced coagulation approximately six-fold more efficiently than the unmodified aptamer. In terms of its anticoagulant activity, the TBA-GLE conjugate approaches NU172, one of the most potent G-quadruplex thrombin aptamers. Molecular dynamics studies have confirmed that the principles applied to the design of the peptide side chain are efficient instruments for improving aptamer characteristics for the proposed TBA conjugate model.

Cytotoxicity reduction by O-nicotinoylation of antiviral 6-benzylaminopurine ribonucleosides.

One of the promising approaches in the development of nucleoside prodrugs is to use the nucleoside analogs containing lipophilic biodegradable residues, which are cleaved to biologically active forms after metabolic transformations in the cell. The introduction of such fragments makes it possible to reduce the general toxicity of the drug candidate and increase its stability in the cell. In order to study the influence of biodegradable lipophilic groups on antiviral activity and cytotoxicity, in this work we synthesized N6-benzyl-2',3',5'-tri-O-nicotinoyl adenosine and N6-(3-fluorobenzyl)-2',3',5'-tri-O-nicotinoyl adenosine, derivatives of N6-benzyladenosine (BAR) and N6-(3-fluorobenzyl)adenosine (FBAR), which had previously shown prominent antiviral activity against human enterovirus EV-A71 but appeared to be cytotoxic. The obtained fully-O-nicotinoylated BAR and FBAR inhibited reproduction of EV-A71 strains BrCr and 46973 and manifested significantly lower cytotoxicity compared to non-protected compounds. In addition, we performed enzymatic hydrolysis of the fully-O-nicotinoylated FBAR in the presence of esterases (CalB and PLE) to investigate metabolic degradation of O-nicotinoylated compounds in cells. Both enzymes hydrolyzed the tested substrate to form the corresponding O-deprotected nucleoside that may suggest the role of hydrolase-type enzymes as general participants of metabolic activation of O-nicotinoylated prodrugs in different cells.

Anticoagulant Oligonucleotide-Peptide Conjugates: Identification of Thrombin Aptamer Conjugates with Improved Characteristics.

Oligonucleotide-peptide conjugates (OPCs) are a promising class of biologically active compounds with proven potential for improving nucleic acid therapeutics. OPCs are commonly recognized as an efficient instrument to enhance the cellular delivery of therapeutic nucleic acids. In addition to this application field, OPCs have an as yet unexplored potential for the post-SELEX optimization of DNA aptamers. In this paper, we report the preparation of designer thrombin aptamer OPCs with peptide side chains anchored to a particular thymidine residue of the aptamer. The current conjugation strategy utilizes unmodified short peptides and support-bound protected oligonucleotides with activated carboxyl functionality at the T3 thymine nucleobase. The respective modification of the oligonucleotide strand was implemented using N3-derivatized thymidine phosphoramidite. Aptamer OPCs retained the G-quadruplex architecture of the parent DNA structure and showed minor to moderate stabilization. In a series of five OPCs, conjugates bearing T3-Ser-Phe-Asn (SFN) or T3-Tyr-Trp-Asn (YWN) side chains exhibited considerably improved anticoagulant characteristics. Molecular dynamics studies of the aptamer OPC complexes with thrombin revealed the roles of the amino acid nature and sequence in the peptide subunit in modulating the anticoagulant activity.

Synthesis of α-D-Ribose 1-Phosphate and 2-Deoxy-α-D-Ribose 1-Phosphate Via Enzymatic Phosphorolysis of 7-Methylguanosine and 7-Methyldeoxyguanosine.

A simple and efficient method for the preparation of α-D-ribose 1-phosphate and 2-deoxy-α-D-ribose 1-phosphate, key intermediates in nucleoside metabolism and important starting compounds for the enzymatic synthesis of various modified nucleosides, has been proposed. It consists in near-irreversible enzymatic phosphorolysis of readily prepared hydroiodide salts of 7-methylguanosine and 7-methyl-2'-deoxyguanosine, respectively, in the presence of purine nucleoside phosphorylase. α-D-Ribose 1-phosphate and 2-deoxy-α-D-ribose 1-phosphate are obtained in near quantitative yields (by HPLC analysis) and 74%-94% yields after their isolation and purification. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of α-D-ribose 1-phosphate barium salt (4a) Alternate Protocol 1: Preparation of 2-deoxy-α-D-ribose 1-phosphate barium salt (4b) Basic Protocol 2: Preparation of α-D-ribose 1-phosphate bis(cyclohexylammonium) salt (5a) Alternate Protocol 2: Preparation of 2-deoxy-α-D-ribose 1-phosphate bis(cyclohexylammonium) salt (5b).